The effect of OH(.) radicals generated by Fenton reaction on the growth and cartilage differentiation in limb bud cell culture.

A consistent chondrogenesis takes place in micro high-density cultures of chick limb bud mesenchyme cells stage 22-24. The effect of an increased generation of OH(.) free radicals by Fenton reaction was tested in these cultures. Components of Fenton reaction (i.e., ferrous iron in form of ADP-Fe2+ complex in 0.1 mM concentration, or 0.2 mM H2O2, or the combination of these components with each other, as well as with 0.2 mM ascorbate) were supplemented to the culture medium after the first 24 h. ADP-Fe2+ complex resulted in a drastic decrease of the frequency and confluence of the cartilage nodules seen in light microscope, accompanied electron microscopically by a strikingly increased frequency of occurrence of lipofuscin-like, residual bodies in the cells. Biochemical methods revealed a significant decrease of both the DNA and glycosaminoglycan contents (to 48.6 and 20.7% of the controls, respectively), in day 6 cultures. H2O2 alone caused similar alterations of the cultures, whereas the combination of it with ADP-Fe2+ complex proved to be lethal for the cells. Ascorbate when added to the ADP-Fe2+-treated cultures displayed a slight protective effect for the glycosaminoglycan content but not for DNA. The results are interpreted in terms of free radical theory of aging.